Signal transduction processes are major targets of drug discovery with G protein-coupled receptors being a primary site of action of many current therapeutic agents. Recent work, however, has shown that signaling pathways are not just linear chains of information but are webs of interacting regulatory molecules in which protein scaffolding, intracellular proximity, and inhibitory control are major determinants of signaling efficacy and specificity. The twenty Regulator of G protein Signaling (RGS) protein family members which inhibit G protein signaling represent a novel site of pharmacologic intervention but: 1) their physiological functions remain incompletely understood and 2) there are no reported small molecule inhibitors of RGS function. The identification of selective RGS inhibitors would provide both: 1) tools for the study of RGS function in cells and in vivo and 2) a starting point for therapeutic drug development. A flow cytometry method for quantitating RGS/Ga interactions will be adapted for multiplexed high-throughput screening for RGS inhibitors. The binding of fluorescently labeled Ga protein subunits to purified RGS proteins on beads will be optimized to permit multiple simultaneous assays of Ga binding to RGS 4, 6, 7, 8, and 9. This multiplexed method will both speed the throughput of screens and will provide immediate information on specificity of the chemicals identified in primary screens. Secondary analyses to demonstrate functional activity and effects in cell culture models are described. The ultimate aim of this project is the identification of selective small molecule inhibitors of RGS action. This will provide important chemical tools and accelerate the development of novel therapeutics.